During tumor progression, tumor cells require more oxygen and metabolites to remove waste products. In addition gaining access to the host vascular system and generation of a tumor blood supply are rate-limiting steps for tumor progression (Bergers et al., 2003). Similarly, diseases such as age-related macular degeneration are accompanied the generation of new blood vessels. (Ng et al., 2006). Thus, tumor blood vessels are prime targets for inhibiting tumor growth and the blood vessels that accompany age-related macular degeneration are suitable targets for inhibiting this disease. Tumor blood vessels express specific markers that are not present in the blood vessels of normal tissues. Many of these specific markers are proteins associated with tumor-induced angiogenesis, the sprouting of new blood vessels (Ruoslahti, 2002). The cell adhesion receptors, integrins αvβ3 and αvβ5, are over-expressed in tumor vasculature (Eliceiri and Cheresh, 1999). Indeed, one of the RGD-peptides identified by in vivo phage-display for tumor homing, or targeting, recognizes αvβ5 (Pasqualini et al., 1997). Peptides specific for these integrins have been used as ligands for targeted delivery of anti-cancer and anti-angiogenic agents (Arap et al., 1998). The heterogeneity in the vasculature might provide new opportunities for targeted delivery of therapies.
Conventional chemotherapy is limited by its toxicity to normal tissues. Therapeutic results might be greatly improved if the chemotherapeutic drugs could bind directly to tumor sites or tumor vessels and be kept away from normal tissues. Most small-molecule chemotherapeutic agents have a large volume of distribution on intravenous (i.v.) administration (Speth et al., 1988). This distribution often leads to a narrow therapeutic index due to a high level of toxicity to normal tissues. Through encapsulation of drugs in a macromolecular carrier, such as liposomes, the volume of distribution is significantly reduced and the concentration of drug in the tumor is increased (Drummond et al., 1999). Encapsulation can decrease the severity and types of nonspecific toxicities and can increase in the amount of drug that is effectively delivered to the tumor site (Gabizon and Martin, 1997; Martin, 1998; Papahadjopoulos et al., 1991). A tumor targeting ligand can be used to target macromolecular carriers, such as liposomes, to the tumor site. Alternatively, drugs can be conjugated or linked to the tumor targeting ligand to facilitate targeting of the drug to the tumor.
Liposomes were suggested as drug carriers in cancer chemotherapy (Gregoriadis et al., 1974). Since then, interest in liposomes has increased and liposome systems are now being extensively studied as drug carriers. Three basic requirements are desired for liposomes for use in specifically delivering drugs: (i) prolonged blood circulation, (ii) sufficient accumulation in tumors or other target tissues, (iii) controlled drug release and uptake by tumor cells or other target tissues with a release profile matching the pharmacodynamics of the drug.
Initially, the research in liposome drug delivery systems suffered from very fast blood clearance by the reticuloendothelial system (RES). It was recognized that particle size, surface charge (Weinstein, 1984), and liposome composition strongly influenced the clearance profile (e.g., incorporation of phosphatidylinositols or monosialogangliosides prolongs liposome circulation in the blood) (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Senior, 1987). This uptake may be evaded by ‘stealth’ liposomes, which preferentially exit the circulation via leaky capillaries and are predicted to accumulate in tumors or other diseased tissue exhibiting extensive neo-vascularisation leading to higher concentrations and enhanced efficacy (Wu et al., 1993).
However, liposomes were only fully recognized as successful drug delivery candidates when it was discovered that liposomes coated with the synthetic polymer polyethyleneglycol (PEG) had significantly increased half-life in the blood (Allen et al., 1991; Blume and Cevc, 1990; Klibanov et al., 1990; Papahadjopoulos et al., 1991; Senior et al., 1991). The pegylated liposomes are long circulating due to a highly hydrated and protected liposome surface, which inhibits protein adsorption and opsonization of the liposomes (Woodle and Lasic, 1992). Having solved the problems of fast opsonization and clearance, providing liposomes with up to 72 h half-life in the blood (Drummond et al., 1999), the next challenge was to get the liposomes to accumulate in the tumor tissue or other diseased tissues through active targeting.
The use of targeting liposomes can potentially lead to significantly enhanced drug release at tumor target sites and increased therapeutic efficacy (Lee et al. 2004; Park et al 2002). The drug delivery research field has successfully constructed long circulating liposomes that accumulate in tumor tissue where the entrapped drugs then leak out of the liposomes by passive diffusion, unless there is an active trigger present. The use of site-specific triggers that can release drugs specifically in diseased tissue is one way of increasing drug bioavailability at the tumor target site. Another way of optimizing drug bioavailability is to obtain a higher degree of liposome accumulation by active targeting. Furthermore, the combination of active targeting with active triggering can potentially lead to significantly enhanced and specific drug release at the tumor target site (Lee et al., 2004; Park et al., 2002).
For solid malignancies, which comprise more than 90% of human cancers, antibodies recognizing tumor-specific antigens have provided little utility for drug delivery since immunoconjugates cannot penetrate tumor tissue (Dvorak et al., 1991; Shockley et al., 1991). However, the development of phage-displayed peptide libraries over the past decade has ushered in the opportunity to identify small peptides that are more effective than antibodies.
Phage-displayed random peptide libraries provide opportunities to map B-cell epitopes (D'Mello et al., 1997; Fu et al., 1997; Scott and Smith, 1990; Wu et al., 2001; Wu et al., 2003) and protein-protein contacts (Atwell et al., 1997; Bottger et al., 1996; Nord et al., 1997; Smith et al., 1999), select bioactive peptides bound to receptors (Koivunen et al., 1999; Li et al., 1995; Wrighton et al., 1996) or proteins (Bottger et al., 1996; Castano et al., 1995; DeLeo et al., 1995; Kraft et al., 1999; Pasqualini et al., 1995), search for disease-specific antigen mimics (Folgori et al., 1994; Liu et al., 2004; Prezzi et al., 1996), and determine cell-specific (Barry et al., 1996; Lee et al., 2004; Mazzucchelli et al., 1999) and organ-specific peptides (Arap et al., 1998; Essler and Ruoslahti, 2002; Pasqualini et al., 1995; Pasqualini and Ruoslahti, 1996).
Screening phage display libraries against specific target tissues would therefore be a direct and fast method in identifying peptide sequences, which are used for targeting of drug or gene delivery vectors. The invention discloses peptides identified by phage display that have therapeutic and diagnostic applications.